Understanding Huntington's Disease

Members of the Wilkinson Lab, wearing red clown noses in support of Red Nose Day, which is a campaign dedicated to raising money for children and young people living in poverty.

Maggie Mang is a Senior in the College double majoring in Biology and Philosophy. She joined the K.D Wilkinson lab in Fall 2014. Maggie is a SIRE Research Partner, SURE alum 2015, SIRE Peer Mentor and SIRE Independent Research Grant Recipient Fall 2015.

For the Fall 2015 semester, most of my research centers on better understanding huntingtin cytotoxicity in Baker’s yeast, Saccharomyces cerevisiae. Huntington’s Disease (HD), which affects about 30,000 people in the United States and has the potential to affect 200,000 more¹, is an incurable, autosomal dominant neurodegenerative disease. HD is categorized by its severe motor, physiological, and cognitive deterioration in patients, and is caused by an expansion of glutamines (poly-Q) within the essential gene, huntingtin (Htt)—the mutation is commonly referred to as Htt103Q. Though the discovery of the genetic basis behind HD happened back in 1993², there has yet to be either a better understanding of the role of Htt or a cure for HD. For anyone who used to watch the popular medical drama, House, one can perhaps remember the storyline of one of the doctors who found out that she had inherited the disease from her mother.

The expansion of these poly-Q stretches within the gene is so deadly because it causes the Htt protein to self-aggregate; these aggregates can then consequentially lead to abnormal protein-protein interactions with other proteins in the cell. It has also been shown that in mammalian cells, the mutant Htt cytotoxicity can also affect effective protein degradation through the endoplasmic reticulum (ER). In mammals, however, this cytotoxicity has been shown to be combated through overexpression of p97/Valosin Containing Protein (VCP), an AAA ATPase chaperone protein³. Similarly, the yeast homolog of p97/VCP, Cdc48, has also been associated with Htt aggregates⁴. In experiments where p97/VCP or Cdc48 have been inhibited, results have included defects in both endocytic sorting and in the degradation of certain proteins⁵. These results point toward an association between p97/VCP and Cdc48.

In yeast and in some mammals, it has been revealed that proteins involved with endocytic machinery are additionally associated with actin filament organization. Proteins such as mammalian WASPs and Las17, its yeast homologue, play essential roles in overall actin filament organization and are, consequentially, implicated in associated endocytic machinery pathways. In yeast, Lsb1 and Lsb2 are Q/N-rich proteins that regulate Las17 activity. It has been previously shown that Q/N-rich proteins can act as either suppressors or enhancers of poly-Q toxicity, depending on the specific cell-cell interactions⁶.

It is interesting to note that cellular interaction of Htt103Q with Lsb2 may have a beneficial effect on the cell. It has been shown that Lsb2 has a role in combating overall cytotoxicity caused by Htt aggregation. Preliminary data indicates that the deletion of Lsb2 protein increases cytotoxicity of mutant Htt while overexpression of Lsb2 decreases mutant Htt cytotoxicity. Other preliminary data also show that the levels of both Lsb1 and Lsb2 are dependent upon the levels of Cdc48.

While initial data shows decreased Htt cytotoxicity by overexpression of Lsb2, the exact mechanism behind how it operates is not yet known. This leads us to consider the role of Cdc48 on Htt toxicity, especially as it relates to the regulation of Q/N-rich proteins.

One of the numerous reasons I am so excited to conduct research into this topic is because it combines research I had done last year through SIRE on Cdc48 and Lsb2 regulation and over this past summer through SURE, on the effect of overexpression of Lsb2 on Htt103Q cytotoxicity. I have found, through my research experiences, that research continually builds off of itself. It sounds like a given in retrospect, but it plays a larger role in research than one immediately realizes.

Last year, when I was working primarily with Cdc48, another member in my lab, Dami, had been working on finding ways to reduce Htt103Q cytotoxicity through over expression of Lsb2. Her initial findings had shown that over expression of Lsb2 reduced cytotoxicity in cells expressing Htt103Q (See image 1). When she graduated and I resumed her research over the summer, I set out to replicate her results (see image 2). However, my results did not show the same trend that Dami’s had originally.

Initially, my lab mentor and I wondered if there were additional factors that we were not taking into account when setting out to replicate these results. After multiple weeks of using these strains, we had zoomed into what we believed was the issue. Dami’s Lsb2 had been tagged with HA (to better ‘tag’ a protein in order to analyze it), while the Lsb2 that I was using had been tagged with mCherry (a red fluorescent tag that’s used to visualize protein whereabouts under a fluorescent microscope). mCherry is a larger tag than that of HA; we hypothesized that the larger size was somehow interfering with some of the molecular mechanisms that HA was not. Going off of this hunch, I redid my previous experiments with the HA tagged Lsb2.

I remember this day very clearly. A few weeks ago, when I took out the plates to see the effect of the HA tagged Lsb2 on Htt103Q cytotoxicity, I was shocked to see the results (see image 3). Compared to the results I got during the summer, the results I had now seemed to be an improvement: the yeast growth was a lot better, and my results matched up with Dami’s earlier results. Just like as seen with Dami’s plates, overexpression of HA-Lsb2 correlated with a decrease in Htt103Q cytotoxicity. My lab mentor and I were so excited about these results that we kept talking about it for the rest of the day—even better, we went into Fall Break absolutely elated at the fact that two people had done the same experiment and, more importantly, had gotten the same results.

In addition, other than looking at the effect of overexpression of Lsb2 on Htt103Q cytotoxicity, I also wanted to see the arrangement of these proteins in the cell itself. To visualize proteins using fluorescence microscopy, I used Htt103Q tagged with GFP, which shows up as bright green, and Lsb2 tagged with mCherry, which shows up as red. From my initial photos, it seemed as if Lsb2 and Htt103 colocalized—as in, they showed up in the same parts of the cell together (see image 4). However, a photograph taken from florescence microscopy can only tell so much—it could not definitively tell us that the two proteins were physically interacting. The photos could only tell us that they were located within proximity to each other—this could also mean that they were on top of each other or next to each other, but did not necessarily mean that they were truly colocalizing.

To help us solve the question of whether they were or were not physically interacting, we sent samples of these cells and proteins to a collaborator (whom another member of our lab, Jennifer, was working with over the summer) who was able to make a 3D representation that corresponds to the physical arrangement of the Htt103Q and Lsb2 proteins in the cell (see image 5). We got the results back in the early part of this semester: The green is Htt103Q, the red is Lsb2, and the yellow corresponds to the actual physical contact between the two proteins. In other words, this 3D reconstruction confirmed what we originally believed was colocalization between these two proteins.

Looking back, it took a long time for our research to arrive at this point. It took many hours, countless experiments and repeats of experiments, and numerous people to reach this stage. Not only did we collaborate with each other—replicating and then building off of each other’s projects—but we also worked with other researchers states away in order to achieve the same end goal. I am even more excited to have a good foundation that shows a relationship between Htt103Q and Lsb2. Moving forward for the remainder of this semester and academic year, I am eager to explore this relationship further, specifically bringing in Cdc48 and seeing its effect on Htt103Q cytotoxicity as well, especially because of its earlier established relationship with Lsb2. This research will not immediately or even directly lead to a cure for HD, but it is one piece in the innumerable number of pieces that, when put together, will constitute a cure for patients living with HD.

If there’s one thing I have learned in the past few months tackling this project, it is that science truly is synergistic but that it takes perseverance to reach this stage of seeing it in retrospect. I am, of course, deeply grateful for the SIRE grant to allow me to continue this research, but also to my immensely supportive lab group who constantly challenges me to be better than who I was yesterday (see image 6).

Image 1

Htt103Q cytotoxicity was measured via a plate assay, in which cells were serially diluted four times. The presence of galactose induces Htt103Q while the presence of copper incudes Lsb2. The SD-ura-leu functions as a control. As shown in the figure, there was a difference in Htt103Q cytotoxicity when in the presence of Lsb2 overexpression. Lsb2 was tagged with HA. (D. Kim, T.A. Chernova, K.D. Wilkinson unpublished data)

Image 2

Htt103Q cytotoxicity was measured via a plate assay, in which cells were serially diluted four times. The presence of galactose induces Htt103Q while the presence of copper incudes Lsb2. The SD-ura-leu functions as a control. As shown in the figure, there was not a noticeable difference between Htt103Q cytotoxicity with and without Lsb2 overexpression. Lsb2 was tagged with mCherry.

Image 3

Htt103Q cytotoxicity was measured via a plate assay, in which cells were serially diluted four times. The presence of galactose induces Htt103Q while the presence of copper incudes Lsb2. The SD-ura-leu functions as a control. As shown in the figure, there was a difference in Htt103Q cytotoxicity when in the presence of Lsb2 overexpression. Lsb2 was tagged with HA.

Image 4

Fluorescence miscropy is used to better visualize proteins that are tagged with a fluorescent tag. Htt103Q was tagged with GFP, which shows up green, and Lsb2 was tagged with mCherry, which shows up red. Photos taken of Htt103Q and Lsb2 and were overlayed on top of each other.

Image 5

Pictures A-C show photos of Htt103Q (green) and Lsb2 (red). A yellow color confirms colocalization between Htt103Q and Lsb2. A shows just the visualization of the proteins, B shows the cells without visualization of the tagged proteins, and C is an overlay of the tagged proteins within the cell itself. Pictures A-C were taken using a confocal microscope while D is a 3D reconstruction of confocal images.

-Maggie Mang

References:

1. http://hdsa.org
2. The Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromo- somes. Cell. 1993;72(6):971–983.
3. Leitman, J., F. Ulrich Hartl and G. Z. Lederkremer (2013). "Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress." Nat Commun 4: 2753.
4. Wang, Y., A. B. Meriin, N. Zaarur, N. V. Romanova, Y. O. Chernoff, C. E. Costello and M. Y. Sherman (2009). "Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery." FASEB J 23(2): 451-463.
5. Meyer, H., M. Bug and S. Bremer (2012). "Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system." Nat Cell Biol 14(2): 117-123.
6. Kayatekin, C., K. E. Matlack, W. R. Hesse, Y. Guan, S. Chakrabortee, J. Russ, E. E. Wanker, J. V. Shah and S. Lindquist (2014). "Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1." Proc Natl Acad Sci U S A 111(33): 12085-12090.